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Endocrinology Vol. 142, No. 7 2820-2832
Copyright © 2001 by The Endocrine Society

ARTICLES

Identification of G Protein-Coupled, Inward Rectifier Potassium Channel Gene Products from the Rat Anterior Pituitary Gland1

Karen A. Gregerson, Thomas P. Flagg2, Thomas J. O’Neill, Mark Anderson, Oanh Lauring, Jill S. Horel2 and Paul A. Welling

Departments of Obstetrics, Gynecology, and Reproductive Sciences (K.A.G., T.J.O., M.A., O.L.) and Physiology (K.A.G., T.P.F., J.S.H., P.A.W.), and the Center for Studies in Reproduction (K.A.G., J.S.H.), University of Maryland, Baltimore, Maryland 21201

Address all correspondence and requests for reprints to: Dr. Karen A. Gregerson, Department of Obstetrics, Gynecology, and Reproductive Sciences, 11–007 Bressler Research Building, University of Maryland, 655 West Baltimore Street, Baltimore, Maryland 21201.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Dopamine (DA) is a physiological regulator of PRL secretion, exerting tonic inhibitory control. DA activates an inward rectifier K+ (IRK) channel in rat lactotropes, causing membrane hyperpolarization and inhibition of Ca2+-dependent action potentials. Both the activation of this effector K+ channel and the inhibition of PRL release are mediated by D2-type receptor activation and pertussis toxin- sensitive G proteins. To study the molecular basis of this physiologically relevant channel, a homology-based PCR approach was employed to identify members of the IRK channel family expressed in the anterior pituitary gland. Nondegenerate primers corresponding to regions specific for IRK channels known to be G protein activated (GIRKs; gene subfamily Kir 3.0) were synthesized and used in the PCR with reverse transcribed female rat anterior pituitary messenger RNA as the template. PCR products of predicted sizes for Kir 3.1, 3.2, and 3.4 were consistently observed by ethidium bromide staining after 16 amplification cycles. The identities of the products were confirmed by subcloning and sequencing. Expression of each of these gene products in anterior pituitary was confirmed by Northern blot analysis.

Functional analysis of the GIRK proteins was performed in the heterologous expression system, Xenopus laevis oocytes. Macroscopic K+ currents were examined in oocytes injected with different combinations of Kir 3.0 complementary RNA (cRNA) and G protein subunit (ß1{gamma}2) cRNA. The current-voltage relationships demonstrated strong inward rectification for each individual and pairwise combination of GIRK channel subunits. Oocytes coinjected with any pair of GIRK subunit cRNA exhibited significantly larger inward K+ currents than oocytes injected with only one GIRK channel subtype. Ligand-dependent activation of only one of the GIRK combinations (GIRK1 and GIRK4) was observed when channel subunits were coexpressed with the D2 receptor in Xenopus oocytes. Dose-response data fit to a Michaelis-Menten equation gave an apparent Kd similar to that for DA binding in anterior pituitary tissue. GIRK1 and GIRK4 proteins were coimmunoprecipitated from anterior pituitary lysates, confirming the presence of native GIRK1/GIRK4 oligomers in this tissue. These data indicate that GIRK1 and GIRK4 are excellent candidate subunits for the D2-activated, G protein-gated channel in pituitary lactotropes, where they play a critical role in excitation-secretion coupling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DOPAMINE (DA) IS a physiological regulator of PRL secretion from the anterior pituitary gland (AP), exerting tonic inhibitory control (1). Dissociation of DA from its receptor appears to be another physiological signal leading to a stimulatory phase of PRL secretion (2). In addition, subnanomolar concentrations of DA have been reported to directly stimulate PRL release (3, 4). This action of very low concentrations of DA is distinct from that initiated by the withdrawal of DA (5). Understanding how one transmitter is able to elicit such diverse responses from the same cell type requires complete elucidation of the effectors involved in the transduction of these signals.

We have identified and characterized a voltage-independent, inwardly rectifying potassium channel activated by DA (KDA) that leads to hyperpolarization of the lactotrope membrane and cessation of calcium-dependent action potentials, the driving force for tonic PRL secretion (6, 7). Both the activation of this effector K+ channel and the inhibition of PRL release are mediated by D2 receptor activation and pertussis toxin (PTx)-sensitive G proteins. In vitro studies from our laboratory have demonstrated a critical role for this KDA channel in both the inhibition of PRL release by physiological (nanomolar) concentrations of DA and the stimulatory phase of PRL secretion elicited by DA withdrawal (5, 8).

The purpose of the present studies was to examine the molecular basis of this physiologically important channel by 1) employing a homology-based PCR approach to identify members of the inward rectifier K+ (IRK) channel family that are expressed in AP, and 2) performing functional analysis of candidate proteins using the Xenopus oocyte expression system. Using this approach we have for the first time cloned G protein-gated IRK channel transcripts from AP tissue. The present report also identifies two subunits, associated in pituitary tissue, which when expressed together exhibit D2 receptor-dependent activation similar to the KDA channel studied in primary lactotropes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
AP tissue and cells
AP cells and tissue were harvested from Sprague Dawley-derived female rats purchased from Charles River Laboratories (Wilmington, MA). All animals were housed and cared for strictly in accordance with USDA regulations and the NIH Guide for the Care and Use of Laboratory Animals in a facility accredited by the American Association for the Accreditation of Laboratory Animal Care. Rats were maintained on a 14-h light, 10-h dark cycle, with food and water available ad libitum. Estrous cyclicity was determined by daily vaginal smears. Animals demonstrating at least two consecutive 4-day cycles were killed on the morning of proestrus, and the APs (with neurointermediate lobes removed) were rapidly dissected free. Tissues for the isolation of RNA or protein were immediately frozen in liquid nitrogen and stored at -80 C. For in vitro studies of lactotropes, pituitary cells were prepared as previously described (8). Briefly, the AP was minced in 2 ml HBSS, then incubated in HBSS containing 0.15% trypsin (Sigma, St. Louis, MO) for 15 min at 37 C. After two washes with Hanks’ Ca2+-, Mg2+-free medium, the tissue fragments were mechanically dispersed by trituration in Hanks’ Ca2+-, Mg2+-free medium, and the cells were separated from any remaining fragments by passing the suspension through a sterile 20-µm pore nylon mesh. Cell yield was quantitated using a hemocytometer, and viability was determined to always be greater than 97% based on trypan blue exclusion.

Reverse hemolytic plaque assay (RHPA)
RHPA was used to identify lactotropes for electrophysiology and for assessment of PRL release from individual cells. Briefly, dissociated pituitary cells (2 x 105 cells/ml) were plated together with an excess of protein A-coated erythrocytes on poly-L-lysine-coated glass coverslips in modified Cunningham chambers (9). The chambers were placed in a 95% air-5% CO2 atmosphere at 37 C for 45 min to allow cells to attach to the coverslip. Unattached cells and excess erythrocytes were rinsed away with DMEM (without phenol red) containing 0.1% BSA (DMEM-BSA). PRL antiserum (arPRL-86; final dilution, 1:200) (10) was then introduced into the chambers and allowed to incubate for 60 min. Chambers were again rinsed, and areas of hemolysis (plaques) surrounding the PRL-secreting cells were initiated with serum complement. When lactotropes were being identified for subsequent electrophysiological studies, the source of complement was the serum harvested from the donor of the pituitary cells (8). This autologous serum was used at a final concentration of 1:140 or 1:160 for 15–20 min. At the end of this incubation, the chambers were dismantled while submerged in DMEM containing 10% deactivated horse serum and 0.04 mg/ml gentamicin, and coverslips with cells attached were transferred immediately to six-well plates containing DMEM with 10% deactivated horse serum and 0.04 mg/ml gentamicin. Cells were maintained in culture for 1–2 days until used for patch-clamp studies.

For analysis of release from individual cells, plaque assays were performed that included 0.5 mM ascorbic acid (control) with or without 10-6 M DA in the incubation with the antiserum. These treatments were also performed in the presence of 10-6 M (+)butaclamol or after pretreatment (8 h) with PTx (1 µg/ml). Complete plaque development was accomplished by incubation with guinea pig serum complement at a final dilution of 1:50 for 30 min (37 C). Cells were then fixed with 2% glutaraldehyde for 10 min on ice, and the cells were stained with phloxine (2.5% in distilled water), followed by 0.05% azure II and 0.02% methylene blue in 0.05% sodium borate buffer. Stains were differentiated in a rosin-ethanol solution to produce bright blue nuclei and pale pink cytoplasm.

Plaque areas were quantitated by morphometric measurements using an image analysis system with JAVA imaging software (Jandel Scientific, Corta Madera, CA). The area of each plaque was calculated from two perpendicular measurements of diameter. Statistical significance was determined by two-way ANOVA followed by a t test between independent means.

Patch-clamp studies
Whole cell membrane potential recordings from plaque-identified lactotropes were made according to the giga-ohm seal patch-clamp technique. The perforated patch modification (11) was used to avoid loss of soluble cytosolic factors. In this configuration, electrical access to the cell interior is obtained through membrane spanning pores formed by antibiotics contained in the patch pipette rather than by disrupting the whole patch membrane. Amphotericin stock solution (6 mg in 0.1 ml dimethylsulfoxide) was made daily, sonicated, and kept on ice. This was diluted in internal recording solution to a final concentration of 200 µg/ml. The tip of the recording pipette was filled with amphotericin-free solution, then back-filled with the amphotericin-containing solution. Access resistances ranged from 6–10 M{Omega}. The standard intracellular solution was comprised of 130 mM potassium aspartate, 20 mM KCl, 10 mM glucose, and 10 mM HEPES. Cells were bathed in standard extracellular solution comprised of 145 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES. All solutions were adjusted to pH 7.3–7.4 and 295–305 mosmol.

Patch-clamp experiments were performed at room temperature in a Lexan recording chamber mounted on the stage of a Nikon Diaphot inverted microscope. A coverslip with the plaque-identified pituitary cells attached comprised the floor of the recording chamber through which external solutions were continuously perfused during the experiment. Application of DA was accomplished by a U-tube device that could be positioned next to a cell to apply and withdraw the test solution rapidly while minimizing mechanical disturbance. The D2 receptor antagonist, (+)butaclamol, was bath applied. Whole cell voltage responses under current clamp were recorded wideband (~50 kHz) on videotape using a digital audio processor interface sampling at 44 kHz (PCM701, Sony, Park Ridge, NJ).

RNA isolation
APs harvested from proestrous female rats were frozen in liquid nitrogen, pooled, weighed, and then homogenized at 5 C in 10 ml of a solution containing 4 M guanidine thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sodium lauryl sarcosine, and 0.1 M ß-mercaptoethanol. Total RNA was subsequently isolated using the extraction procedure described by Chomczynski and Sacchi (12). Cerebellar and cardiac atrial RNA were harvested in an identical manner. Both were then selected for polyadenylated [poly(A)+] RNA by oligo(deoxythymidine) chromatography using either spin columns (Pharmacia Biotech, Piscataway, NJ) or the PolyAttract system (Promega Corp., Madison, WI). Yields of messenger RNA (mRNA) were 1–2% of the total RNA with OD 260/280 ratios greater than 1.80. RNA integrity was assessed by denaturing agarose gel electrophoresis. In some studies isolation of AP mRNA was performed directly from tissue homogenates using the FastTrack 2.0 system according to the manufacturer’s recommendations (Invitrogen, San Diego, CA) with similar results.

RT-PCR
Rat pituitary gland mRNA (2–5 ng) was reverse transcribed using oligo(deoxythymidine) and SuperScript reverse transcriptase (200 U) at 42 C for 50 min using the SuperScript RT kit (Life Technologies, Inc.). After the RT reaction, ribonuclease H was added to each reaction tube (0.1 U/ml) and incubated at 37 C for 20 min. Negative control reactions (RT-) were handled in an identical manner, except that reverse transcriptase was excluded.

Nondegenerate primers corresponding to regions specific for IRK channels known to be G protein activated (GIRKs; gene subfamily Kir 3.0) were synthesized and used in the PCR with reverse transcribed female rat AP mRNA as the template. Each PCR reaction was carried out in 50 µl containing 1 µl AP RT reaction solution, 50 pM 5'- and 3'-primers, and reagents from the GeneAmp PCR Reaction system (Perkin-Elmer Corp., Palo Alto, CA) including 1 U AmpliTaq DNA polymerase. The hot start method was used to initiate the reaction. AmpliTaq DNA polymerase was withheld until the reaction temperature had reached 75 C. After the addition of the enzyme, the reaction was sequentially cycled 36 times for 1-min durations at each of the following temperatures: 94 C (denaturing); 54.5 C (Kir 3.2), 57 C (Kir 3.1 and Kir 3.4), or 62.5 C (Kir 3.3) (annealing); and 74 C (extending), using a thermal cycler MJ Research, Inc. (Cambridge, MA). All reactions were incubated at 72 C for 5 min after the last cycle for final extension, then placed on ice.

Kir 3.0 isoform-specific primer sets were used to independently and specifically amplify Kir 3.0 complementary DNAs (cDNAs) that are expressed in the AP. The subunit-specific primer design shown with respect to the proposed primary structure of Kir channels is illustrated in Fig. 4Go. The primers used were oligonucleotides corresponding to base pairs as follows: for Kir 3.1, bp 378–401 (sense, 5'-ATGTCGGCAACTACACTCCCTGTG-3'; melting temperature (Tm) = 61.9 C) and bp 1115–1092 (antisense, 5'-CCTGCTCTTTCACACTGTACGGAG-3'; Tm = 61.9 C); for Kir 3.2, bp 569–586 (sense, 5'-CGGGGAGATATGGACCAC-3'; Tm = 49.5 C) and bp 1123–1105 (antisense 5' CAGTTCCTCTTTAGGCAGC 3'; Tm = 46.6 C); for Kir 3.3, bp 511–528 (sense, 5'-TCGACCTGGAGCACCTGG-3'; Tm = 54 C) and bp 1313–1293 (antisense, 5'-GGGGATGGACCAGTAGAGATG-3'; Tm = 52.3 C); and for Kir 3.4, bp 522–541 (sense, 5'-TCCGAGGTGATCTGGACCAC-3'; Tm = 58.7 C) and bp 1302–1282 (antisense, 5'-CCATTCCTCTTCATTTCTGCC-3'; Tm = 55.2 C). The specific sequences were chosen because these primers correspond to regions that are not well conserved across all of the known Kir channels (0–65% identity at the nucleotide level).



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  		Figure 4. G protein-gated K+ channel subunits are expressed in the anterior pituitary gland. A, Subunit-specific primer design is shown with respect to the proposed primary structure of Kir channels. {blacksquare}, The two membrane-spanning domains that flank a putative pore region. The predicted sizes of the amplified products are shown. B, RT-PCR products, after 36 amplification cycles, using the Kir 3.0-specific primers and AP mRNA as a template. Products were resolved by agarose gel electrophoresis and ethidium bromide staining.

 
For absolute identification, PCR products were size fractionated by agarose gel (1.2%) electrophoresis, excised, extracted, subcloned into the SrfI site of pCR-Script, and sequenced in the Biopolymer Laboratory at University of Maryland (Baltimore, MD). Both strands of each subcloned PCR product was sequenced by automated ABI Prism dye terminator cycle sequencing (PE Applied Biosystems, Inc., Foster City, CA). Oligonucleotides corresponding to the T3 or T7 site of the plasmid pCR-Script or internal sites of Kir 3.2 were used.

Analysis of cycle-dependent amplification was used to determine the relative differences in mRNA levels for the various GIRK isoforms expressed in AP tissue. PCR reactions were carried out as described above, except the number of cycles varied, ranging from 16–30. The same AP RT+ reaction was used as the template for amplification of all the unknown PCR reactions. To ensure that the various primer sets amplified with similar efficiencies, identical cycle-dependent PCR reactions were run using a known quantity of the subcloned Kir 3.0 cDNAs as the templates for amplification (Fig. 4BGo). Each PCR reaction volume (50 µl) contained 0.5 pg of the clone in the pCR-Script vector. The PCR products were fractionated by electrophoresis in a 0.8% agarose gel, stained in ethidium bromide, visualized with a UV transilluminator, and photographed using type 665 positive/negative film (Polaroid Corp., Cambridge, MA). The negative images of the amplified products were analyzed by autoradiographic scanning using a model 620 video densitometer (Bio-Rad Laboratories, Inc., Richmond, CA). The amount (band intensity x area) of an amplified product was calculated as the area under each sample band after baseline subtraction.

Northern blot analysis
Equal amounts (10 µM) of poly(A)+ RNA extracted from APs and atria of proestrous rats were separated on 1.2% agarose-formaldehyde gels and transferred to nylon membrane. 32P-Labeled random prime extension probes corresponding to the regions amplified in the RT-PCR analyses (see Fig. 4Go) were generated for each Kir 3.0 isoform. The hybridization was performed in 50% formamide hybridization buffer at 42 C. Final washes were of high stringency [0.1 x SSC (standard saline citrate) and 0.1% SDS, 50 C]. Blots were exposed to Kodak XAR-5 film (Eastman Kodak Co., Rochester, NY) with an intensifying screen at -70 C.

Complementary RNA (cRNA) synthesis
RNA was transcribed in vitro in the presence of capping analog [m7G(5')ppp(5')G] using the mMESSAGE mMACHINE kit (Ambion, Inc., Austin, TX). Briefly, linearized cDNA was used as the template with the appropriate RNA polymerase (T7 or SP6) used in two reactions. After deoxyribonuclease treatment, cRNA was purified by phenol-chloroform extraction and ammonium-acetate/ethanol precipitation. Yield and concentration were quantified by spectrophotometry.

Oocyte injection
Standard protocols for the isolation and care of Xenopus laevis oocytes were followed. Briefly, frogs were anesthetized by immersion in 0.5% tricaine, and a partial oophorectomy was performed through an abdominal incision. Oocyte aggregates were manually dissected from the ovarian lobes and then incubated in Ca2+-free ORII medium (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES, pH 7.5) containing collagenase (Sigma, type IA, 2 mg/ml) for approximately 2 h at room temperature to remove the follicular layer. After washing the oocytes extensively with collagenase-free ORII, they were placed in L15 medium (50% Leibovitz’s medium and 10 mM HEPES, pH 7.5) and stored at 19 C. Twelve to 15 h after isolation, healthy-looking Dumont stage V–VI oocytes were pneumatically injected (PV 820 picopump, WPI, New Haven, CT) with 50 nl water containing 0–5 ng cRNA. This range was chosen for optimal expression based on our previous studies. The oocytes were then stored in L15 medium at 19 C, and channel activity was assessed 2–6 days postinjection. X. laevis donors were selected for those bearing oocytes with high translation activity and low endogenous Kir channel activity (13).

Two-microelectrode voltage clamp
Whole cell oocyte currents were monitored using a two-microelectrode voltage clamp equipped with a bath clamp circuit (OC-725B, Warner, New Haven, CT). For these studies, oocytes were placed in a small lucite chamber and continually superfused with Ca-free ND88 (88 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 5 mM HEPES, pH 7.4) or KD-90 solution (90 mM KCl, 1 mM MgCl2, and 5 mM HEPES, pH 7.4) at room temperature (21-23 C). Voltage-sensing and current-injecting electrodes had resistances of 0.5–1.5 M{Omega} when back-filled with 3 M KCl. After attaining a stable impalement such that both electrodes measured the same spontaneous membrane potential (±4 mV), the pulse protocols shown below were conducted. Stimulation and data acquisition were performed with a Macintosh Centris 650 computer using an Instrutech ITC16 A/D, D/A converter and Pulse software. Data were filtered at 2 kHz and digitized on-line to the hard disk using Pulse for later analysis using Pulsefit.

Immunoprecipitation and Western blot analysis
APs, cerebellum, and cardiac atria harvested from proestrous female rats were frozen in liquid nitrogen until used for protein analysis. Tissues were homogenized in solubilization buffer [50 mM HEPES (pH 7.6), 150 mM NaCl, 1% Triton X-100 with 1 mM EGTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonylfluoride] at a concentration of approximately 20 mg/ml. After 30 min on ice, lysate was cleared by centrifugation (10 min at 10,000 x g), and the supernatant was saved.

For Western blots, the cleared lysate was mixed 1:1 with 2 x Laemmli sample buffer and boiled for 5 min. After SDS-PAGE, protein was transferred to nitrocellulose (Schleicher & Schuell, Inc., Keene, NH) and blocked overnight with 2% nonfat milk/Tris-buffered saline. Blots were exposed to primary antibodies for 1 h. The anti-GIRK1 antibody (Alomone Laboratories, Jerusalem, Israel) was used at a final concentration of 0.4 ng/ml. The anti-GIRK4 antibody (aCIRN2) (14) was used at a final concentration of 1.2 ng/ml. Both of these antibodies have been extensively characterized and demonstrated to be specific. Indeed, the anti-GIRK4 antibody generated by Krapivinsky provided the critical reagent for identification of the GIRK4 protein in cardiac atrium. Chemiluminescent detection was performed with ECL (Amersham Pharmacia Biotech, Arlington Heights, IL).

For immunoprecipitations, the cleared lysate was generally diluted 1:1 with wash buffer (50 mM HEPES, 150 mM NaCl, and 0.1% Triton X-100). To 1 ml of this diluted lysate, 3–5 µl of primary antiserum were added. Samples were incubated at 4 C for 1 h with constant rocking. Fifty microliters of 50% protein A-agarose (Sigma) were added, and the incubation was continued for an additional hour. Samples were washed four times with 1 ml wash buffer (50 mM HEPES, 150 mM NaCl, and 0.1% Triton X-100) and boiled in 125 µl 1 x Laemmli sample buffer. Generally, 35 µl of this final sample were loaded in a lane for SDS-PAGE and processing for Western blots as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DA inhibits the release of PRL from individual AP cells, as illustrated in Fig. 1Go and summarized in Fig. 2Go. During a 1-h incubation, 10-6 M DA was able to suppress spontaneous PRL release, as represented by plaque area, by approximately 67% (Figs. 1BGo and 2Go). Consistent with the pharmacology of the D2 subtype of receptors, this effect of DA was blocked by 10-6 M (+)butaclamol, a selective D2 antagonist (Figs. 1CGo and 2Go), whereas the inactive isomer (-)butaclamol (10-6 M) did not block DA inhibition of PRL release (data not shown). DA inhibition of PRL release was also blocked by pretreatment of the pituitary cells with pertussis toxin (an ADP-ribosylating agent that functionally uncouples members of the Gi/Go G protein family (Figs. 1DGo and 2Go). As shown in Fig. 3Go, the DA-activated hyperpolarization of individual lactotropes exhibits identical pharmacology as the inhibitory effects on PRL release: D2 receptor subtype and PTx sensitivity. The D2 antagonist, (+)butaclamol, blocked the DA-activated K+ current in 6 of 6 cells, whereas application of (-)butaclamol (4 cells) was without effect. The DA-induced hyperpolarization was absent in all lactotropes pretreated with PTx (8 cells), whereas we routinely found a robust response in greater than 85% of lactotropes (6) (11 of 12 cells in the present study). Pretreatment of cells with cholera toxin (6 cells) was without effect on the hyperpolarization elicited by DA (Fig. 3Go), demonstrating that the activation of KDA is independent from the effects of DA on cAMP in pituitary lactotropes.



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  		Figure 1. Low magnification, darkfield photomicrographs of RHPA illustrate the pharmacology of dopaminergic inhibition of PRL release. A, Spontaneous PRL release was assayed in control medium (0.5 mM ascorbic acid in DMEM). B, Plaque formation in the presence of 10-6 M DA. C, Plaque formation during incubation with 10-6 M (+)butaclamol and 10-6 M DA. D, Plaque formation in the presence of 10-6 M DA by cells pretreated with PTx (1 µg/ml; 8 h). Calibration bar, 300 µm.

 


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  		Figure 2. Summary of RHPA, demonstrating pharmacology of dopaminergic inhibition of PRL release from individual cells. PRL release was assayed for 60 min in the absence () or presence of 10-6 M DA ({blacksquare}). Some assay groups included 10-6 M (+)butaclamol in the incubation medium [(+)butacl]. Other assay groups used anterior pituitary cells that had been incubated with PTx (1 µg/ml) for 8 h before being subjected to RHPA (PTx). *, P < 0.001 compared with the control group without DA.

 


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  		Figure 3. Pharmacology of the DA-activated hyperpolarization in identified PRL-secreting cells. In all experiments 10-6 M DA was applied to the cell via a U-tube device during the time period indicated by the bars. The bath solution (standard extracellular solution) in the recording chamber was continuously exchanged throughout the experiments. A, Rapidly activated, long-lasting hyperpolarization induced by DA. B, Bath application of 10-6 M (+)butaclamol (10 min, interrupted trace) blocked the DA-induced hyperpolarization. Note the increased membrane conductance (as indicated by the reduced membrane potential responses to injected hyperpolarizing current pulses) during the first application of DA. In the presence of (+)butaclamol, DA failed to elicit either a hyperpolarization of the cell or a change in membrane conductance. C, This cell was pretreated with PTx (1 µg/ml) for approximately 8 h before cell recording. DA failed to elicit either a hyperpolarization of the cell or a change in membrane conductance. D, Lactotropes pretreated with ChTx (50 µM, 60 min) still responded to DA with a robust hyperpolarization.

 
Using a homology-based PCR approach, rat AP tissue was found to express transcripts belonging to the G protein-coupled subfamily (Kir 3.0, or GIRK) of the IRK channel family (Fig. 4Go). Figure 4AGo illustrates the Kir 3.0 subunit- specific primer design and predicted amplification product size with respect to the proposed primary structure of Kir channels (shaded bar). The filled boxes represent the two membrane-spanning domains that flank the pore region of the channel. PCR products of predicted sizes for Kir 3.1, 3.2, and 3.4 were observed by ethidium bromide staining after 16 amplification cycles (n = 3 for each transcript). A PCR product of predicted size for Kir 3.3 was also obtained, but required many more amplification cycles to produce a detectable product. In contrast, Kir 3.3 product was easily amplified when reverse transcribed cerebellum mRNA from the same animal was used as the template (not shown). The products for each Kir 3.0 subunit obtained after 36 PCR amplification cycles are shown in Fig. 4BGo. No products were observed in reactions using mRNA rather than first strand cDNA as the template, ruling out spurious genomic amplification (Fig. 4BGo). The identities of the PCR products were confirmed by subcloning and sequencing several independent clones. Sequences were identical to the corresponding regions published for Kir 3.1, 3.2, 3.3, and 3.4.

The relative abundance of the four Kir 3.0 transcripts in AP tissue were assessed by cycle dependence analysis. In these studies the amount of PCR product produced from AP first strand cDNA (AP RT+) was determined for a number of consecutive amplification cycles for each transcript and compared with the cycle-dependent amplification of a known amount of Kir 3.0 cDNA (Fig. 5AGo). These data fit the expected characteristics of PCR amplification in which the amount of products produced initially increases exponentially, but then the rate of production slows and levels off (plateau effect) (15). Because the cycle dependence of the cDNA amplifications is identical for each isoform (Fig. 5BGo), the amplification efficiency of each different primer set is not significantly different. Therefore, differences in the cycle-dependent amplification of products from the AP-RT+ template (upper series of photographs in Fig. 5AGo) can be used to estimate relative differences in the expression levels of the various Kir 3.0 transcripts in AP tissue. Although the relative abundance of Kir 3.1, 3.2, and 3.4 in AP tissue is quite similar, expression levels of Kir 3.3 are 2–3 orders of magnitude less (Fig. 5CGo).



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  		Figure 5. Cycle-dependent PCR amplification of Kir 3.0 transcripts from anterior pituitary mRNA. A, PCR products generated from increasing number of amplification cycles and using Kir 3.0-specific primers. Either reversed transcribed AP (AP RT) mRNA (upper row of gel photographs) or the specific subcloned Kir 3.0 cDNA (lower row of gel photographs) was used as the template for amplification. B, PCR amplification curves for the products generated from each Kir 3.0 cDNA and the corresponding specific primer set. Curves were generated by plotting the log of the measured PCR product (intensity) vs. the amplification cycle number (n = 3). Note that the curves are superimposable, indicating that the efficiencies of the various primers sets are not different. C, Abundance of Kir 3.0 mRNAs expressed in AP relative to Kir 3.1. The relative abundance was determined from the linear phase of the amplification curves of products from AP RT template.

 
Expression of the three more abundant gene products in proestrous AP tissue was confirmed by Northern blot analysis (Fig. 6Go). The Kir 3.1 probe hybridized to transcripts of 4.5 and 6.0 kb in both AP and cardiac atrial mRNA. (Transcripts of 4.5 and 6.0 kb in atria also reported in Refs. 14 and 16). The Kir 3.2 probe hybridized to a 2.4-kb transcript in AP only, consistent with reports that this isoform is not expressed in heart (17). A 2.4- to 2.8-kb transcript for Kir 3.2 in human insulinoma cells has been reported (18). The Kir 3.4 probes hybridized to transcripts of 3.6 and 5.1 kb in both AP and atria.



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  		Figure 6. Northern blot analysis confirms the presence of Kir 3.1, Kir 3.2, and Kir 3.4 mRNA in anterior pituitary tissue. Equal amounts (10 µg) of poly(A)+ RNA extracted from APs and atria of proestrous rats were separated on 1.2% agarose-formaldehyde gel, transferred to nylon membrane, and hybridized with 32P-labeled random prime extension probes. Final washes were of high stringency (0.1 x SSC, 0.1% SDS, 50 C).

 
Functional analysis of these GIRK proteins was performed in the heterologous expression system, X. laevis oocytes. In the first series of studies, macroscopic K+ currents were examined in oocytes injected with different combinations of GIRK cRNA and G protein subunit (ß1{gamma}2) cRNA. Representative families of K+ currents in oocytes injected with GIRK1, GIRK2, GIRK4, or pairwise combinations of each are shown in Fig. 7AGo. The current-voltage relationships demonstrate strong inward rectification for each individual and pairwise combination of GIRK channel subunit (Fig. 7BGo), characteristic of the Kir channel family. Oocytes coinjected with any pair of GIRK subunit cRNA exhibit significantly larger inward K+ currents than oocytes injected with only one GIRK channel subtype (Fig. 7CGo). These data are consistent with the theory that the G protein-gated Kir channels are heteromultimers.



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  		Figure 7. G protein-gated Kir channels (GIRKs) expressed in the anterior pituitary gland are heteromultimers. Macroscopic K+ currents in X. laevis oocytes injected with different combinations of GIRK cRNA and G protein subunit (ß1{gamma}2) cRNA. A, Representative families of K+ currents in oocytes injected with GIRK1, GIRK2, GIRK4, or pairwise combinations of each. Oocytes were held at 0 mV in 90 mM K and stepped in 20 mV increments from -150 to +50 mV. B, Current-voltage relationships demonstrate strong inward rectification for each individual and pairwise combination of GIRK channel subunit. C, Oocytes coinjected with any pair of GIRK subunit cRNA exhibit significantly larger inward K+ currents than oocytes injected with only one GIRK channel subtype (currents at -90 mV are normalized to the average GIRK2 current and compared, mean ± SD; by ANOVA, P < 0.0001). Each group was injected with an identical amount of GIRK cRNA.

 
Ligand-dependent activation of only one of the GIRK combinations (GIRK1 + GIRK4) was observed when channel subunits were coexpressed with the D2 receptor in Xenopus oocytes (see Table 1Go). GIRK1 and GIRK4 were coexpressed with either the long (D2L, Fig. 8Go) or the short (D2S, Fig. 9Go) form of the D2 receptor. Representative currents, measured in the presence and absence of a maximal dose of DA (10 µM), are shown in Figs. 8AGo and 9AGo. The addition of DA significantly increased channel activity (Figs. 8BGo and 9BGo), although the presence of considerable ligand-independent current suggests significant G protein-coupled receptor precoupling. Dose-response data fit to the Michaelis-Menten equation (solid line) gave an apparent Kd of 11.07 ± 2.14 nM for D2L (Fig. 8CGo) and 17.58 ± 7.78 nM for D2S (Fig. 9CGo). However, it should be noted that most of the data points obtained were at the minimum or maximum response level.


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  		Table 1. IRK currents in oocytes coexpressing D2 receptor and GIRK combinations

 


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  		Figure 8. Macroscopic K+ currents in X. laevis oocytes coexpressing GIRK1, GIRK4, and the long form of the D2 receptor (D2L). A, Representative K+ currents, measured in the presence and absence of a maximal concentration of DA (10 µM). Oocytes were held at 0 mV in 90 mM K and stepped in 20-mV increments from -130 to +50 mV. B, DA (10 µM) significantly increased GIRK1/GIRK4 channel activity (by paired t test, P < 0.001), although considerable ligand-independent channel activity was observed. Currents at -90 mV are compared (mean ± SD). C, Dose-response data (0, 10, 25, 50, 100, 500, and 1000 nM) fit to the Michaelis-Menten equation (solid line) with an apparent Kd of 11.07 ± 2.14 nM.

 


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  		Figure 9. Macroscopic K+ currents in X. laevis oocytes coexpressing GIRK1, GIRK4, and the short form of the D2 receptor (D2S). A, Representative K+ currents, measured in the presence and absence of a maximal concentration of DA (10 µM). Oocytes were held at 0 mV in 90 mM K and stepped in 20-mV increments from -130 to +50 mV. B, DA (10 µM) significantly increased GIRK1/GIRK4 channel activity (by paired t test, P < 0.01), although considerable ligand-independent channel activity was observed. Currents at -90 mV are compared (mean ± SD). C, Dose-response data (0, 10, 25, 50, 100, 500, and 1000 nM) fit to the Michaelis-Menten equation (solid line) with an apparent Kd of 17.58 ± 7.78 nM.

 
To determine whether native GIRK1 and GIRK4 proteins are present and do associate in AP cells, these proteins were immunoprecipitated from pituitary membranes. Anti-GIRK1 immunoprecipitated and labeled a broad band between 67K and 72K (Fig. 10AGo, lane 3), which represents heavily glycosylated GIRK1 as also found in cardiac atrial membranes (14). This mature form of the GIRK1 protein coimmunoprecipitated with GIRK4 as shown in lane 4 of Fig. 10AGo and lane 6 of Fig. 10CGo. Likewise, GIRK4 coimmunoprecipitated with GIRK1, as evidenced by the faint band at 45K recognized by the anti-GIRK4 (Fig. 10AGo, lane 1). This 45K GIRK4 is seen more clearly in lane 2 when anti-GIRK4 was used for immunoprecipitation. The immunoprecipitation with anti-GIRK1 was repeated using a lower concentration of detergent (0.325% vs. 1% Triton X-100, final concentration), which improved recovery of the associated GIRK4 protein (Fig. 10BGo, lane 4). As shown in Fig. 10BGo, GIRK4 coimmunoprecipitated with GIRK1 from both AP (lane 4) and atrial (lane 3) tissue, but not cerebellar (lane 5) tissue where GIRK4 is not expressed. The 45K GIRK4 protein is also clearly seen in Western blot analysis of whole cell lysates from AP and atrium (Fig. 10BGo, lanes 1 and 2). The large band migrating at 50K in the immunoprecipitated samples is the heavy chain of the rabbit antibodies used for the immunoprecipitations. This is verified in the controls presented in Fig. 10CGo. Staining of the heavy chain is shown in lanes 1 and 5, which were only loaded with the anti-GIRK1 antiserum (no cell lysate). Also contributing to this band is any contaminating protein A Sepharose as demonstrated in lanes 2 and 8, which contained cell lysate processed for immunoprecipitation with protein A-Sepharose, but without any precipitating antibodies. This large band at 50K (arrowheads) obscures the immature, nonglycosylated GIRK1 protein seen in whole cell lysate of AP tissue (arrow, lane 4, Fig. 10CGo) and has been previously reported in cardiac atria (14). To further establish that the coimmunoprecipitations of the GIRK1 and GIRK4 proteins truly reflect their association in AP membranes and were not a procedural artifact, antiserum against the Kir 2.1 channel (Alomone Laboratories, Jerusalem, Israel) was used for immunoprecipitation. This inwardly rectifying K+ channel does not assemble with GIRK proteins (19), and the anti-Kir 2.1 did not coprecipitate either GIRK1 or GIRK4 (Fig. 10CGo, lanes 3 and 7, respectively) from AP membranes.



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  		Figure 10. Native GIRK1 and GIRK4 proteins are associated in anterior pituitary tissue. A, Proteins immunoprecipitated from AP cell lysate were subjected to Western blot using anti-GIRK4 (lanes 1 and 2) or anti-GIRK1 (lanes 3 and 4). Antiserum to GIRK1 immunoprecipitated GIRK4 as well as GIRK1 (lanes 1 and 3). Anti-GIRK4 immunoprecipitated the same pair of pituitary proteins (lanes 2 and 4). The predicted sizes of mature, glycosylated GIRK1 (67–72 kDa) and GIRK4 (45 kDa) are indicated by the arrows. B, Western blot of proteins using anti-GIRK4. Immunoprecipitation with anti-GIRK1 was repeated with whole cell lysates prepared from AP, cardiac atrium (At), and cerebellum (Cb). GIRK4 coimmunoprecipitated with GIRK1 from atrial and pituitary (lanes 3 and 4), but not from cerebellum membranes (lane 5). GIRK4 was also recognized in Western blot of whole cell lysates (wcl) from both atrium and AP (lanes 1 and 2). C, Western blot controls using anti-GIRK1 (lanes 1–4) or anti-GIRK4 (lanes 5–8). Antibody only (lanes 1 and 5) or cell lysates processed with protein A-Sepharose without precipitating antibodies (lanes 2 and 8) show staining of a 50K band (arrowheads). A similar sized band stains in anterior pituitary whole cell lysate and is the predicted size of nonglycosylated GIRK1 (arrow, lane 3). Anti-Kir 2.1 did not immunoprecipitate either GIRK1 (lane 3) or GIRK4 (lane 7). The immunoprecipitation procedure used in A differed from that used in B and C in the final concentration of Triton X-100 (1% vs. 0.325%, respectively). The band migrating at 50 kDa in the gels presented in A and B and marked by the arrowhead (hc) is the heavy chain of the rabbit Igs used for the immunoprecipitations (compare with lanes 1 and 5 in C).

 
Antibodies to GIRK2 are also available from Alomone Laboratories. However, under these same conditions, repeated attempts to visualize GIRK2 on Western blots failed, even when using cerebellum, which is known to express the GIRK2 protein.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Pituitary expression of Kir 3.1 (GIRK1) has been demonstrated with in situ hybridization (20) and immunocytochemistry (21). The present findings establish that multiple forms of G protein-gated, inwardly rectifying K+ channels are expressed in the AP of the rat. Through molecular cloning, semiquantitative PCR, functional reconstitution studies, and coimmunoprecipitation analysis we have also identified a GIRK1/GIRK4 heteromultimer as the most logical candidate for the DA-activated K+ channel in lactotropes.

Molecular characterization of the G protein-gated IRKs in pituitary tissue is a necessary step in elucidating the physiological role of the DA-activated K+ channel in the regulation of PRL secretion. Dopaminergic inhibition of PRL release is mediated via the D2 subtype of DA receptors (22, 23). D2 receptors are both physically and functionally coupled to a PTx-sensitive G protein-coupled receptor (24, 25), and this receptor-G protein coupling plays a necessary role in the dopaminergic inhibition of secretion (26, 27, 28). In vitro studies demonstrate that a DA-activated K+ conductance, exhibiting identical pharmacological properties as these, plays a critical role in mediating the effects of DA on PRL release (5).

We have identified and characterized a voltage-independent K+ channel underlying this DA-activated conductance (KDA). Activation of this channel involves the D2 subtype of DA receptor and a PTx-sensitive G protein-coupled receptor and occurs over the same range of DA concentrations that inhibit PRL release (5–100 nM) (6). Single channel analysis has demonstrated that the KDA is a 40pS K+ channel that is voltage independent and inwardly rectifying (7). These are the two hallmarks of the IRK channel family (gene nomenclature Kir) (29, 30). The Kir 3.x subfamily of the IRK channels can be directly activated by G proteins (GIRKs) as is the KDA channel (31).

The DA-activated K+ conductance we have described is independent of Ca2+ and membrane potential and thus can be activated at or near the normal resting potential of the cell to impact on its physiology. This is consistent with experimental findings that a major action of DA in lactotropes is to activate a K+ conductance that is not normally manifested in the absence of DA and can support a long-lasting (at least many minutes) hyperpolarization (6, 7). Furthermore, these data indicate that members of the Kv channel family that are present in lactotropes in the absence of DA (32) and require membrane depolarization or Ca2+ for activation do not underlie the DA-activated responses.

Several experimental findings are consistent with the hypothesis that D2 receptors in lactotropes are coupled to the IRK channel directly and are not mediated by soluble cytoplasmic factors. First, we found that the DA-activated hyperpolarizing response is not lost over periods up to 1 h or more of whole cell dialysis during electrophysiological studies. Second, the DA-activated hyperpolarization is not diminished by buffering both external and internal Ca2+ (with 10 mM EGTA and 10 mM BAPTA, respectively) (6). In addition, the robust electrical responses are independent of D2 regulation of adenylate cyclase, as demonstrated in perforated patch recordings from lactotropes treated with cAMP-elevating agents (33). Finally, single D2 agonist-activated K+ channels have been recorded in outside-out patches that are identical to those observed in cell-attached patches in both conductance and gating properties (31). These data suggest that the D2 receptor-K+ channel coupling in lactotropes is mediated through a membrane-delimited pathway. Members of the G protein gated subfamily of IRK channels (Kir 3.0) can be directly activated by Gß{gamma} dimers in a manner consistent with such membrane-delimited activation of native channels (34, 35).

The expression of Kir 3.1, 3.2, and 3.4 in the AP make these proteins candidate subunits of the DA-activated, IRK channel in lactotropes. The abundance of these three transcripts in AP tissue was quite similar, and these transcripts were readily detected using Northern blot analysis of AP mRNA. Although we were able to amplify specific GIRK3 product from AP tissue, the low abundance of this transcript puts into question its physiological relevance.

The results of our functional analysis of the other three GIRK channel subunits are consistent with current theory that the G protein-gated Kir channels function as heteromultimers (14, 36). Oocytes injected with any pairwise combination of GIRK subunit cRNA always exhibited significantly larger inward K+ currents than oocytes injected with only one of the GIRK channel subtype pair. Injection of GIRK2 alone did produce K+ currents larger than either GIRK1 alone, GIRK4 alone, or the GIRK1/GIRK4 pair, as has been shown previously (36), and there is evidence that functional channels are formed from GIRK2 homomultimers (37).

Coexpression of the GIRK1/GIRK4 subunit pair with either the long (D2L) or the short (D2S) form of the D2 receptor resulted in ligand-dependent activation of the inward rectifying K+ current in Xenopus oocytes. The AP contains the mRNAs for both D2 receptor isoforms, although the expression levels of D2L are considerably greater than those of D2S (38, 39). DA dose-response data of the GIRK1/GIRK4 combination, fit to the Michaelis-Menten equation, yielded an apparent Kd of 11.07 ± 2.14 nM for D2L or 17.58 ± 7.78 nM for D2S, consistent with the Kd for ligand binding to native DA receptors in AP tissue (22) and within the concentration-dependent range of DA activation of the native KDA channel in identified lactotropes (6).

No ligand-dependent activation of either GIRK1/GIRK2 or GIRK2/GIRK4 was observed when these subunit pairs were coexpressed with either D2 receptor form. These findings suggest that the channels formed by these pairs are not functionally coupled to the D2 receptor. However, it is also possible that the very large ligand-independent currents produced by GIRK2 expression alone and in combination with other subunits masked any further increase in response to DA. Injecting smaller quantities of channel mRNA (to decrease channel and current density) did not reveal ligand activated currents in GIRK2 combinations (data not shown). Kuzhekandathil et al. (37) reported ligand-activated current when D2 receptors are coexpressed with GIRK2 homomultimers in CHO cells. Others (40, 41) have reported coupling between DA receptors and channels when only GIRK1 mRNA was injected. However, these channels were probably heteromultimers of the GIRK1 with the endogenous Kir channel found in Xenopus oocytes (XIR) (42, 43). In our studies we selected for oocytes expressing low endogenous Kir channel activity and did not examine D2 coupling with currents in oocytes injected with only one GIRK subunit mRNA. Further arguing against GIRK2 as a critical component of the lactotrope DA-activated channel is that PRL regulation appears normal in the weaver (wv) mutant mouse (44). The wv phenotype is associated with a missense mutation in the pore region of GIRK2 (45), rendering the channel constitutively active and nonselective in ion permeation. Homozygote mice have normal serum and pituitary contents of PRL, and the normal sex difference (PRL higher in females) is intact.

The presence of considerable ligand-independent GIRK1/GIRK4 channel activity in both D2L- and D2S-injected oocytes may be due to significant precoupling between the receptors and the GTP-binding proteins. This ligand-independent channel activity was consistently greater in the presence of the D2S receptor form. The two isoforms of the D2 receptor result from alternative splicing of the D2 DA receptor gene with the D2L isoform, including an insertion of 29 amino acids in the putative third cytoplasmic domain (38, 46, 47) that has been shown to be important for the coupling of seven-transmembrane receptors to GTP-binding proteins (48, 49) and differences in the efficiency of the two D2 isoforms to activate various effectors. For example, the D2S isoform has been demonstrated to be more efficient than D2L in inhibiting adenylate cyclase activity in JEG3 cells (50). The difference between the two receptor isoforms has been suggested to confer differential coupling to various G protein subtypes. Thus, the difference in ligand-independent GIRK1/GIRK4 current between D2L- and D2S-injected oocytes may reflect the specific G protein subtypes endogenously expressed in Xenopus oocytes. Studies to elucidate the apparent receptor-G protein precoupling are underway.

D2 receptor-dependent activation of GIRK1/GIRK4 is similar to the KDA channel in primary lactotropes. GIRK1 and GIRK4 have been demonstrated to be the subunits comprising the muscarinic-activated K+ channel in heart (14). Although many of our previous and present findings parallel those reported for the atrial KACh channel, important differences exist between the two native channels. First, the KDA channel in pituitary lactotropes exhibits weaker inward rectification in whole cell recordings, and although the atrial channel exhibits both rapid activation and rapid deactivation upon application and withdrawal of acetylcholine (51), the DA-induced hyperpolarization of lactotropes lasts considerably beyond agonist withdrawal (31). Such differences can reflect tissue-specific posttranslational processing of the subunit proteins, tissue-specific expression of different GTP-binding proteins, and/or differential involvement of additional signal transduction pathways that modulate the activity of the receptor-G protein-gated channel. In addition, the coexpression of other Kir subfamily members in the AP, which may interact with the GIRK proteins is possible. For example, Kir 2.3 has been cloned from GH3/B6 cells, a mammosomatotrope cell line, and demonstrated by Northern blot analysis to be expressed in AP tissue (52). Clearly, complete analysis of IRK subunits in AP tissue must be performed.

One must also consider the heterogeneity of cell types in this gland. Lactotropes are only one of six different secretory cell types in the AP. A recent report indicates that thyrotropes express GIRK1 (21), and GH-secreting adenoma cells isolated from acromegalic patients can express a DA-activated K+ current (53). In corticotropes, a constitutively active IRK current inhibited by CRH has been reported (54), as has evidence for a glybenclamide-sensitive component of proadrenomedullin N-terminal 20 peptide inhibition of ACTH release (55). However, in proestrous female glands, which were used in the present study, nearly half of the AP cells are lactotropes (10), and nearly 90% of lactotropes exhibit functional expression of the DA-activated K+ channel (6). Nevertheless, further molecular characterization of the DA-activated K+ channel in normal lactotropes will require cellular localization of specific Kir transcripts.

In conclusion, we demonstrate that three different G protein-gated K+ channel subunits are expressed in the AP gland: GIRK1, GIRK2, and GIRK4 (Kir 3.1, Kir 3.2, and Kir 3.4). Functional analyses of these Kir channel subunits in Xenopus oocytes demonstrate that the coexpression of GIRK1 and GIRK4 with the D2 receptor produces a DA-activated channel whose properties recapitulate many of the properties of the native KDA channel in pituitary lactotropes. Moreover, we found that native GIRK1/GIRK4 oligomers exist in AP membranes. Molecular characterization of the KDA channel is a critical step in elucidation of the pleiotropic actions of DA in the regulation of lactotrope function and PRL secretion.


   Acknowledgments
 
We are grateful to the following people for their generous gifts of cDNAs: Dr. Olivier Civelli (through Dr. Paul Albert) for D2L and D2S; Dr. David Clapham for GIRK4 (CIR); and Dr. Henry Lester for GIRK1 and Gß1{gamma}2. Thanks also to Dr. Grigory Krapivinsky for his generous gift of anti-GIRK4 (aCIRN2).


   Footnotes
 
1 This work was supported by USPHS Grants DK-40336 (to K.A.G.) and DK-48271 (to P.A.W.). Back

2 Supported by a predoctoral fellowship on an interdepartmental NIH Training Program in Integrative Membrane Biology (T32-GM-08181). Back

Received December 27, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Ben-Jonathan N 1985 Dopamine: a prolactin-inhibiting hormone. Endocr Rev 6:564–589[CrossRef][Medline]
  2. Martinez de la Escalera G, Weiner RI 1992 Dissociation of dopamine from its receptor as a signal in the pleiotropic hypothalamic regulation of prolactin secretion. Endocr Rev 13:241–255[CrossRef][Medline]
  3. Denef C, Manet D, Dewals R 1980 Dopaminergic stimulation of prolactin release. Nature 285:243–246[CrossRef][Medline]
  4. Burris TP, Stringer LC, Freeman ME 1991 Pharmacological evidence that a D2 receptor subtype mediates dopaminergic stimulation of prolactin secretion from the anterior pituitary gland. Neuroendocrinology 54:175–183[Medline]
  5. Gregerson KA, Golesorkhi N, Chuknyiska R 1994 Stimulation of prolactin release by dopamine withdrawal: role of membrane hyperpolarization. Am J Physiol 267:E781–E788
  6. Gregerson KA, Einhorn L, Smith MM, Oxford GS 1989 Modulation of potassium channels by dopamine in rat pituitary lactotrophs: a role in the regulation of prolactin secretion? In: Oxford GS, Armstrong CM (eds) Secretion and Its Control. Rockefeller University Press, New York, pp 123–141
  7. Einhorn LC, Gregerson KA, Oxford GS 1991 D2 dopamine receptor activation of potassium channels in identified rat lactotrophs: whole-cell and single-channel recording. J Neurosci 11:3727–3737[Abstract]
  8. Ho M-Y, Kao JPY, Gregerson KA 1996 Dopamine withdrawal elicits prolonged calcium rise to support prolactin rebound release. Endocrinology 137:3513–3521[Abstract]
  9. Cunningham AJ, Szenberg A 1968 Further improvements in the plaque technique for detecting single antibody-forming cells. Immunology 14:599–601[Medline]
  10. Gregerson KA 1995 Complement action on secretory cells identified by the reverse hemolytic plaque assay: modified assay eliminates exposure of secretory cells to complement. Endocrine 3:371–376
  11. Horn R, Marty A 1988 Muscarinic activation of ionic currents measured by a new whole-cell recording method. J Gen Physiol 92:145–159[Abstract/Free Full Text]
  12. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  13. Welling PA 1997 Primary structure and functional expression of a cortical collecting duct Kir channel. Am J Physiol 273:F825–F836.
  14. Krapivinsky G, Gordon EA, Wickman K, Velimirovic B, Krapivinsky L, Claphan DE 1995 The G-protein-gated atrial K+ channel IKAch is a heteromultimer of two inwardly rectifying K+-channel proteins. Nature 374:135–141[CrossRef][Medline]
  15. Erlich HA, Gelfand D, Sninsky JJ 1991 Recent advances in the polymerase chain reaction. Science 252:1643–1651[Abstract/Free Full Text]
  16. Kubo Y, Reuveny E, Slesinger PA, Jan YN, Jan LY 1993 Primary structure and functional expression of a rat G-protein-coupled muscarinic potassium channel. Nature 364:802–806[CrossRef][Medline]
  17. Lesage F, Duprat F, Fink M, Guillemare E, Coppola T, Lazdunski M, Hugnot J-P 1994 Cloning provides evidence for a family of inward rectifier and G-protein coupled K+ channels in the brain. FEBS Lett 353:37–42[CrossRef][Medline]
  18. Ferrer J, Nichols CG, Makhina EN, Salkoff L, Bernstein J, Gerhard D, Wasson J, Ramanadham S, Permutt A 1995 Pancreatic islet cells express a family of inwardly rectifying K+ channel subunits which interact to form G-protein-activated channels. J Biol Chem 270:26086–26091[Abstract/Free Full Text]
  19. Tinker A, Jan YN, Jan LY 1996 Regions responsible for the assembly of inwardly rectifying potassium channels. Cell 87:857–868[CrossRef][Medline]
  20. Karschin C, Schreibmayer W, Dascal N, Lester H, Davidson N, Karschin A 1994 Distribution and localization of a G protein-coupled inwardly rectifying K+ channel in the rat. FEBS Lett 348:139–144[CrossRef][Medline]
  21. Morishige K, Inanobe A, Yoshimoto Y, Kurachi H, Murata Y, Tokunaga Y, Maeda T, Maruyama Y, Kurachi Y 1999 Secretagogue-induced exocytosis recruits G protein-gated K+ channels to plasma membrane in endocrine cells. J Biol Chem 274:7969–7974[Abstract/Free Full Text]
  22. Caron MG, Beaulieu M, Raymond V, Gagne B. Drouin J, Lefkowitz R, Labrie F 1978 Dopaminergic receptors in the anterior pituitary gland. J Biol Chem 253:2244–2253[Free Full Text]
  23. Enjalbert A, Bockaert J 1983 Pharmacological characterization of the D2 dopamine receptor negatively coupled with adenylate cyclase in rat anterior pituitary. Mol Pharmacol 23:576–584[Abstract]
  24. Kilpatrick BF, Caron MG 1983 Agonist binding promotes a guanine nucleotide reversible increase in the apparent size of the bovine anterior pituitary dopamine receptors. J Biol Chem 258:13528–13534[Abstract/Free Full Text]
  25. Senogles SE, Benovic JL, Amlaiky N, Unson C. Milligan G, Vinitsky R, Spiegel AM, Caron MG 1987 The D2-dopamine receptor of anterior pituitary is functionally associated with a pertussis toxin-sensitive guanine nucleotide binding protein. J Biol Chem 262:4860–4867[Abstract/Free Full Text]
  26. Cronin MJ, Myers GA, MacLeod RM, Hewlett EL 1983 Pertussis toxin uncouples dopamine agonist inhibition of prolactin release. Am J Physiol 244:E499–E504
  27. Enjalbert A, Mussett F, Chenard C, Priam M, Kordon C, Heisler S 1988 Dopamine inhibits prolactin secretion stimulated by the calcium channel agonist Bay-K-8644 through a pertussis toxin-sensitive G protein in anterior pituitary cells. Endocrinology 123:406–412[Abstract]
  28. Schofield JG, Khan AI, Wood A 1988 Modification by pertussis toxin of the responses of bovine anterior pituitary cells to acetylcholine and dopamine: effects on hormone secretion and 86Rb efflux. J Endocrinol 116:393–40128[Abstract/Free Full Text]
  29. Aldrich R 1993 Advent of a new family. Nature 362:107–108[CrossRef][Medline]
  30. Doupnik CA, Davidson N, Lester HA 1995 The inward rectifier potassium channel family. Curr Opin Neurobiol 5:268–277[CrossRef][Medline]
  31. Einhorn LC, Oxford GS 1993 Guanine nucleotide binding proteins mediate D2 dopamine receptor activation of a potassium channel in rat lactotrophs. J Physiol 462:563–578[Abstract/Free Full Text]
  32. Lingle CJ, Sombati S, Freeman ME 1986 Membrane currents in identified lactotrophs of rat anterior pituitary. J Neurosci 6:2995–3005[Abstract]
  33. Gregerson KA D2 receptor activation of a K+ conductance in lactotropes is independent of D2 inhibition of adenylate cyclase activity. 77th Annual Meeting of The Endocrine Society, Washington DC, 1995 (Abstract P1-1)
  34. Logothetis DE, Kurachi Y, Golper J, Neer EJ, Clapham D 1987 The ß{gamma}-subunit of GTP-binding proteins activate the muscarinic K+ channel in heart. Nature 325:321–326[CrossRef][Medline]
  35. Reuveny E, Slesinger PA, Inglese J, Morales JM, Iniguez-Lluhi JA, Lefkowitz RJ, Bourne HR, Jan YN, Jan LY 1994 Activation of the cloned muscarinic potassium channel by G protein ß{gamma} subunits. Nature 370:143–146[CrossRef][Medline]
  36. Kofuji P, Davidson N, Lester HA 1995 Evidence that neuronal G-protein-gated inwardly rectifying K+ channels are activated by Gß{gamma} subunits and function as heteromultimers. Proc Natl Acad Sci USA 92:6542–6546[Abstract/Free Full Text]
  37. Kuzhikandathil EV, Yu W, Oxford GS 1998 Human dopamine D3 and D2L receptors couples to inward rectifier potassium channels in mammalian cell lines. Mol Cell Neurosci 12:390–402[CrossRef][Medline]
  38. Dal Toso R, Sommer B, Ewert M, Herb A Pritchett DB, Bach A,. Shivers BD, Seeburg PH 1989 The dopamine D2 receptor: two molecular forms generated by alternative splicing. EMBO J 8:4025–4034[Medline]
  39. Montmayeur J-P, Bausero P, Amlaiky N, Maroteaux L, Hen R, Borrelli E 1991 Differential expression of the mouse D2 dopamine receptor isoforms. FEBS Lett 278:239–243[CrossRef][Medline]
  40. Werner P, Hussy N, Buell G, Jones KA, North RA 1996 D2, D3, and D4 dopamine receptors couple to G protein-regulated potassium channels in Xenopus oocytes. Mol Pharmacol 49:656–661[Abstract]
  41. Pillai G, Brown NA, McAllister G, Milligan G, Seabrook GR 1998 Human D2 and D4 dopamine receptors couple through ß{gamma} G-protein subunits to inwardly rectifying K+ channels (GIRK1) in a Xenopus oocyte expression system: selective antagonism by L-741,626 and L-745,870 respectively. Neuropharmacology 37:983–987[CrossRef][Medline]
  42. Hedin KE, Lim NF, Clapham DE 1996 Cloning of a Xenopus laevis inwardly rectifying K+ channel subunit that permits GIRK1 expression of IKACh currents in oocytes. Neuron 16:423–429[CrossRef][Medline]
  43. Kennedy ME, Nemec J, Corey S, Wickman K, Clapham DE 1999 GIRK4 confers appropriate processing and cell surface localization to G-protein-gated potassium channels. J Biol Chem 274:2571–2582[Abstract/Free Full Text]
  44. Schwartz NB, Szabo M, Verina T, Wei J, Dlouhy SR, Won L, Heller A, Hodes ME, Ghetti B 1998 Hypothalamic-pituitary-gonadal axis in the mutant weaver mouse. Neuroendocrinology 68:374–385[CrossRef][Medline]
  45. Patil N, Cox DR, Bhat D, Faham M, Myers RM, Peterson AS 1995 A potassium channel mutation in weaver mice implicates membrane excitability in granule cell differentiation. Nat Genet 11:126–129[CrossRef][Medline]
  46. Giros B, Sokoloff P, Martres M-P, Riou J-F, Emorine LJ, Schwartz JC 1989 Alternative splicing direct the two D2 dopamine receptor isoforms. Nature 342:923–926[CrossRef][Medline]
  47. Monsma FJ, McVittie LD, Gerfen CR, Mahan SC, Sibley DR 1989 Multiple D2 receptors produced by alternative RNA splicing. Nature 342:926–929[CrossRef][Medline]
  48. Kobilka BK, Kobilka TS, Daniel K, Regan JW, Caron MG, Lefkowitz RJ 1988 Chimeric {alpha}2-ß2 adrenergic receptors: dilineation of domains involved in effector coupling and ligand binding specificity. Science 240:1310–1316[Abstract/Free Full Text]
  49. Lechleiter J, Hellmiss R, Duerson K, Ennulat D, David N, Clapham D, Peralta E 1990 Distinct sequence elements control the specificity of G protein activation by muscarinic acetylcholine receptor subtypes. EMBO J 9:4381–4390[Medline]
  50. Montmayeur J-P, Borrelli E 1991 Transcription mediated by a cAMP-responsive promoter element is reduced upon activation of dopamine D2 receptors. Proc Natl Acad Sci USA 88:3135–3139[Abstract/Free Full Text]
  51. Breitwieser GE, Szabo G 1988 Mechanism of muscarinic receptor-induced K+ channel activatio